Swimming. Pools. TREECE, Ph.D., F.A.P.H.A. a central Streptococcus fecalis type and. wide distribution have been noted.'16-8

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1 Nov., 1948 Sanitary Significance of Cocci in Swimming. Pools CASSANDRA RITTER, F.A.P.H.A. AND E. LEE TREECE, Ph.D., F.A.P.H.A. Division of Sanitation, Kansas State Board of Health, Lawrence; and Department of Bacteriology, University of Kansas, Lawrence, Kan. T HERE has long been a feeling among sanitarians and laboratory workers that the test for the coliform group of organisms is not an adequate index of the safety of swimming pool water, since this index does not take into consideration the possibility of contamination from the skin and upper respiratory tract of the bather. The streptococci have been suggested and investigated as indicators of pollution. Mallmann and associates 1-4 proposed a method of isolation, and they and others 5 gave evidence to show that these organisms fluctuate with the bathing load and are resistant to chlorine. Standard Methods for the Examination of Water and Sewage6 in the 8th and earlier editions gave tentative and optional directions for the determination of streptococci by making microscopic examinations of the sediment from lactose-broth fermentation tubes. In the 9th edition, no reference is made to streptococci in the section on bacteriological control of swimming pools. The use of Neisseria catarrhalis as an index of pollution has been proposed by Tapley and Jennison.7 The isolation of Staphylococcus albus from swimming pools was reported by Stokes,8 but it has not been considered as an indicator organism. Methods for classifying the streptococci have been outlined by Sherman.9 The normal fecal streptococci were studied by many workers,-15 and classed as [1532] a central Streptococcus fecalis type and variants, some undoubtedly salivary types; and some strains similar to Streptococcus pyogenes. The low invasive powers of the enterococcus and its wide distribution have been noted.'16-8 The incidence of various types of streptococci in the nose and throat has been determined under diverse conditions.'19-2 Better methods for the isolation of the streptococci have become available recently through the use of sodium azide as an inhibitory agent for Gram-negative rods. It was used in a 1-5,000 concentration by Mallmann 22 for the detection of streptococci in sewage, and by other workers26' 27 for the isolation of enterococci from various waters. The present authors devised a new liquid medium containing sodium azide for use in this survey. The study here reported was made to determine the incidence of streptococci in swimming pools in Kansas during the summer seasons; to correlate this information with incidence of coliform organisms and the sanitary data; and to classify the streptococci isolated, and determine their resistance to chlorine. Tests were made during the summers of 1942 and At those times the junior author was engaged in the inspection of all pools in Kansas while employed by the State Board of Health, thus affording an opportunity for the collection of samples under actual operating conditions. The data collected

2 Vol. 38 can be compared as *to type of pool, equipment and operation, chlorine residual and ph at the time the sample was collected, and bacteriological quality. In the course of the routine inspection of the pools, samples of water were collected and shipped to the State Water and Sewage Laboratory where colony counts and the determination of coliform organisms were made according to Standard Methods.6 At the pool-side, samples to determine the presence of streptococci were taken by adding ml. of the water to a bottle containing ml. of double strength sodium azide medium. It immediately neutralized the free or combined available chlorine that might be present. The medium has the formula: Per cent Proteose peptone No Dextrose NaCl SWIMMING POOLS Sodium azide ph Sterilize in the autoclave It supported a luxuriant growth of all the strains of streptococci and staphylococci tested. They included the hemolytic strains Lancefield's Groups A, B, C, D, E, F, G; Streptococcus viridans; S. salivarius, S. fecalis, Staphylococcus aureus, and S. Albus. With samples of swimming pool water and of similar mixed flora, coliform organisms and other rod forms did not develop. Thb medium has been found useful for the isolation of streptococci and staphylococci from a variety of sources, such as from waters both heavily'polluted and of good quality, and from skin, nasal, and throat swabs. For use in the survey here reported, the medium was prepared in double strength. Bottles inoculated in the field were shipped or carried to the laboratory where they were incubated at 370 C. Growth was indicated by turbidity and appeared in 1 to 3 days in 80 per cent 1533 of the cases, rarely as late as 7 days. Negative samples remained clear and were incubated from to 14 days before being discarded. Samples showing growth in the sodium azide medium were streaked on plates of proteose No. 3 hemoglobin agar, from which colonies were picked for further study. Only Gram-positive cocci developed in the sodium azide medium. In the majority of cases, there was one strain of streptococcus alone; occasionally there were two strains, and in 33 cases out of 7 examinations of turbid samples a staphylococcus was present either alone or with a streptococcus. Contaminations, which were from spore-formers, were rare and were observed orily after prolonged inctibation of the primary sample. Ninety-two different pools were inspected and 179 samples collected during two summers. Forty-nine pools were the fill-and-draw type, which is generally unsatisfactory from a sanitation standpoint as it is difficult to maintain the water in good physical condition and with a constant chlorine residual. Forty-three were recirculating pools, which give better opportunity for control of water quality. The difference in pool type was clearly reflected in the bacteriological analyses. Of 88 samples from fill-and-draw pools, 28, or 31.8 per cent, showed the presence of coliform organisms; of 91 samples from recirculating pools, only 4, or 4.4 per cent, showed coliforms. For streptococci, of the 88 samples from fill-and-draw pools, 55, or 62.5 per cent, were positive as contrasted with 22, or 24.2 per cent, of the samples from recirculating pools. For staphylococci the corresponding figures were 6.8 and 29.7 per cent respectively. Coliform organisms were present 36.4 per cent of the time when streptococci were recovered, and 'only 3.9 per cent of the time when they were not found. Staphylococci were isolated 27 times

3 AMERICAN JOURNAL OF PUBLIC HEALTH 1f534 Nov., 1948 from pools negative for streptococci and a stool culture; S. salivarius from saliva 6 times from streptococcus-positive samples. They might have been present and division isolated from a normal throat. and another organism of the viridans overgrown or overlooked in other streptococcus-positive samples. Table 1, in which strains showing simi- The results of these tests are shown in No chlorine was present at the time lar characteristics are grouped together. of collection of the sample in 67.5 per Morphologically the organisms isolated in the survey were Gram-positive cent of those showing streptococci and in only 23.5 per cent of the samples cocci grouped as pairs and in short negative for these organisms. In the chains. The biological characteristics cases with chlorine present, orthotolidine class them as enterococci. Of the 79 readings showed that free available strains studied, 52 showed biological reactions typical of S. zymogenes and S. chlorine was present in amounts varying from 0.05 p.p.m. to 0.8 p.p.m., or combined available chlorine in amounts, ations from the control enterococcus fecalis. Twenty-six strains showed vari- from 0.4 p.p.m. to 0.8 p.p.m. cultures as follows: 3 strains did not Certain of the pools are outstanding grow at 0 C. and 6 at 45 C.; 6 did for excellence of equipment and operation and are eligible for special certifi- would not tolerate 6.5 NaCl; 16 failed not develop in alkaline medium; 9 cation by the rules of the State Board to grow in 0.1 per cent methylene blue, of Health. The results of 26 analyses and 23 were negative for strong reduction. from 11 certified pools showed that the pool w.ater was in acceptable condition, Studies of hemolytic type were interesting but of no great value in classifica- as coliform organisms were not found; residual chlorine was present each time, tion. In poured plates of horse blood although only times did it reach the agar, no colonies showing the beta type required 0.4 p.p.m.; and the ph was in of hemolysis were observed among the the correct range. Yet streptococci were cultures isolated. Seventy-two of the found 3 times and staphylococci 8 times 79 strains produced the prime in the 26 tests. type of hemolysis. They exhibited considerable variation as to width and com- In studying the streptococci recovered, use was made of the tests outlined by pleteness of the hemolytic zone produced, width and darkness of the zone Sherman9 as determining.the primary divisions of the genus. They include of red blood cells around the colony, determinations of hemolytic type by time of appearance of the characteristics, and amount of change during the Brown's28 method; growth at 450 C. and 0 C.; in broth containing 6.5 per succeeding periods of incubation and refrigeration. Seven cultures showed cent sodium chloride; in broth adjusted to ph 9.6; in milk containing 0.1 per hemolysis; the 6 of them classed as cent methylene blue; and in milk to observe strong reduction of dye. The for biological characteristics. enterococci were in the groups atypical serological grouping by the Lancefield29 All of the cultures grew abundantly precipitin test was determined for a part on proteose No. 3 hemoglobin agar. of the cultures, as was colony type on Strains typical of S. fecalis formed agar containing 5 per cent sucrose. As opaque colonies 1 mm. in diameter. A a basis of comparison for the study, five smaller number of strains from the atypical group produced a distinct green known cultures were included: Streptococcus pyogenes and S. zymogenes coloration, differing in amount and in which were Lancefeld Group A and time of appearance. Group D respectively; S. fecalis from The precipitin reaction by the

4 Vol. 38 SWIMMING POOLS 1535 Control Cultures Strep. pyogenes Strep. salivarius Viridans Division Strep. zymogenes Strep. fecalis Cultures from pools No. of Strains Enterococcus Div. I II 2 III 4 IV 4 1 V S VI Viridans Division 1 Characteristics beta beta prime prime cs pime TABLE 1 of Streptococci Isolated from Swimming Pools Growth at Growth in Presence of f- A '1% t~~~ Cs "C I w- %0 r. 0.2 bo z0e v G 0 -M C.r O~~~~~~~~ c'n C; ~~~~~o z =)u a 0.0 (/2 :~ A D D '+ ± + + +* ± t t t _ + + _ _ a 01 0 '0 la 0 a D -. D & K D & G G & K None - z None None Lancefield technique was determined for 33 of the cultures. Of these, 27 were typical enterococci and read as follows: 17 Group D; 7 cross-reactions for Groups D and K; 1 for Groups D and G; 1 for Groups G and K; and 1 gave no precipitate. Six of the atypical strains were tested and gave no precipitate. Only 1 strain showed the reactions of a streptococcus of the viridans division. It produced the type of hemolysis, grew at 450 but not at C., reduced methylene blue slowly, was negative in the tolerance tests, and did not produce mucoid colonies on 5 per cent sucrose agar. No strains of S. salivarius were found in the group studied. That this organism was recovered. from three pools is suggested by preliminary tests of cultures that did not survive storage long enough to be classified. Later studies of 17 samples from a single recirculating indoor pool yielded S. salivarius twice and atypical enterococcus strains three times. In many of the 179 analyses, chlorine residuals were adequate. Streptococci were isolated once from water having 0.5 p.p.m. free chlorine and a ph under 6.8; once with 0.4 p.p.m. residual and ph 7.6; and once with 0.8 p.p.m. combined residual chlorine and ph 7.6. Staphylococci were found in the presence

5 AMERICAN JOURNAL OF PUBLIC HEALTH 1536 Nov., 1948 TABLE 2 Minutes Required to Produce 0 Per cent Kill of Various Bacteria by Different Amounts of Free Available Chlorine at Room Temperature and ph 7.0 to 7.2 Control Cultures Beta hemolytic Streptococci: Group A Group D Strep. jecalis Strep. salivarius Viridans Division Stapk. 209 Esch. coli A. aerogenes Cultures from Pools Typical Enterococci I Atypical Enterococci II IV 1. VI > > > > >180 > > Free available chlorine in p.p.m > > S so S 5 of 0.5 p.p.m. and 0.1 p.p.m. free chlorine, and 0.8 p.p.m. combined chlorine. The resistance of various organisms to free available chlorine is important in swimming pool control. Therefore, chlorine tolerance tests were made for 11 of the strains of streptococcus isolated from pools in this. study, 5 control strains of streptococcus and 1 strain each of Escherichia coli and Aerobacter aerogenes, isolated trom untreated surface water, and of Staphylococcus 209, the F.D.A. strain used for testing disinfectants. Tests were made in chlorinefree, chlorine-demand-free water after the method of Butterfield and coworkers,31 and the free available chlorine was checked by the orthotolidine-arsenite method of Hallinan.32 In Table.2 are presented the results of tests made at room temperature in distilled water buffered to a ph of 7.0 to 7.2. Comparing a series in which the chlorine content was 0.6 p.p.m. at the beginning of the experiment and not less than 0.4 p.p.m. at the end, it is shown that under those conditions the control strains of beta hemolytic streptococci, Group A and Group D, were killed almost immediately. The control strain of S.- fecalis survived 1 hour but not 1Y2 hours. Of 7 similar strains isolated from pools, 3 survived an hour or more and only 1 was killed in less than 5 minutes. The 4 atypical enterococcus strains survived from 5 to minutes. S. salivarius survived 5 minutes but not minutes; another control strain of the viridans group survived minutes. Staphylococcus 209 survived minutes. E. coli and A. aerogenes were killed almost immediately, none surviving 5 minutes' exposure. A more limited experiment of the survival of these organisms in chlorinefree sterilized tap water showed that a Group A streptococcus survived 1 %4 but not 4 hours. S. salivarius survived for 4 but not 7 hours. The enterococci, both typical and atypical strains, survived 12 hours or longer, 1 strain being present after 4 days. Tlhree strains of

6 Vol. 38 SWIMMING POOLS 1537 staphylococci survived for 7 but not 12 hours. A later study on a recirculating pool demonstrated the fact that proper chlorination will control the organisms present. From a sample collected one morning containing no chlorine, coliforms, staphylococcus, and 1 strain each of S. salivarius, and an atypical enterococcus were isolated. Chlorination was started and by the time the free residual chlorine had reached 0.4 p.p.m., the streptococci and coliforms, had disappeared. The pool was in use during the day; staphylococci were isolated at intervals, but disappeared after a longer period of chlorination. The presence of coccus forms on the skin of the potential bathers was also investigated. Thirty-one cultures from 18 persons were made by rubbing small areas in the cubital, axillary, perineal, and inguinal regions and the abdominal area with moist sterile swabs which were then incubated in tubes of sodium azide broth, from which isolations were subsequently made. Staphylococci were recovered from every swab (37 strains). Streptococci were recovered every time from the perineal region and 3 times, with 5 strains, from swabs from the inguinal region. Of the 22 streptococcus strains thus isolated, could be classified as S. fecalis, as atypical enterococci, and 2 as S. salivarius. None of the strains of staphylococci' isolated showed the characteristics of pathogenic strains. SUMMARY AND CONCLUSIONS It is noteworthy that no streptococci of the beta hemolytic type were isolated in this study. That they will grow readily in the primary medium used was demonstrated in the laboratory. Their low resistance to free available chlorine indicates that they would survive but a short time after being introduced into a properly chlorinated pool. Of 79 strains of streptococci isolated from swimming pools, 52, or 65.8 per cent, can be classified as Streptococcus fecalis. Their ability to grow under adverse conditions-provided by high and low temperatures, alkaline medium and high salt content-seem consistent with their high rate of survival. This is also shown by their resistance to chlorine, as strains picked at random were resistant to 0.4 to 0.6 p.p.m. free available chlorine from minutes to 1 hour, under the conditions of the test. Twenty-six strains were similar to S. fecalis in only part of their characteristics. They may be considered as more closely related to the enterococci than to any other division of the streptococci. Tests for the temperature limits of growth and for growth in alkaline medium were found to be the most reliable in classifying the enterococci. Typical S. fecalis strains were classified as Type D by the Lancefield technique; atypical strains could not be typed. That Streptococcus salivarius may be present in swimming pool water would be expected. No strains were recovered in this survey, although their presence was suggested by the preliminary tests from three pools. This organism was shown to be killed by 0.4 to 0.6 p.p.m. free available chlorine in minutes or less. In 179 analyses from 92 swimming pools, streptococci were recovered 83 times, and coliform organisms 32 times. Pools equipped for recirculation and continuous chlorination of the water were shown to be superior to pools of the fill-and-draw type from the viewpoint of water quality. From the results of this work, it can be concluded that a standard requiring the absence of streptococci from swimming pool water is more stringent than that requiring the absence of coliform organisms. And it is our opinion, based on public health experience, that this additional requirement is not necessary.

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